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NIFK / MKI67 FHA domain-interacting nucleolar phosphoprotein · IHC design guide
Design Immunohistochemistry for NIFK
Plan NIFK paraffin IHC around its nucleolar tissue pattern (HPA tissue IHC). Compare high staining in colon glandular cells with undetected staining in adipocytes (HPA tissue IHC), and start with the catalog antibody’s 1:50–1:200 IHC dilution range (datasheet: A04797).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NIFK Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Nucleolar staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread nucleolar staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across tissue samples (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium RNA concordance (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | No isoforms reported; no membrane topology (UniProt) |
Section 1
Recommended NIFK IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published NIFK IHC workflow using hospital samples (PMC10553176 methods).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A04797) |
| Fixation | Image fixative and duration unreported (datasheet A04797); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-NIFK, 1:50-1:200 (datasheet A04797) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NIFK-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nucleolar expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule; UniProt Q9BYG3: nuclear localisation).
Section 2
What Is the Expected NIFK Staining Pattern?
NIFK is a nuclear protein concentrated in nucleoli and also localizes to mitotic chromosomes (UniProt Q9BYG3 subcellular location). In paraffin sections, expect nucleolar staining in many cell types, including strongly stained colon glandular cells and cerebral cortex neurons (HPA: ubiquitous nucleolar expression; High in both examples). Interpret intensity by cell type: HPA rates its tissue IHC profile Supported, with medium consistency between staining and RNA data (HPA: reliability). NIFK has no transmembrane segment (UniProt Q9BYG3 topology).
| Discrete nuclear dots in colon glandular cells or cerebral cortex neurons. | This fits the expected nucleolar pattern in cell types scored High by tissue IHC (HPA: tissue IHC). Judge staining within identifiable cells against the counterstained nucleus; the tissue-level label does not mean every cell must stain equally (HPA: cell-specific ratings). |
| Predominantly membrane or broad cytoplasmic staining, without convincing nucleolar signal. | Treat this as a suspect pattern: NIFK is nucleolar and lacks a transmembrane segment (UniProt Q9BYG3 subcellular location and topology). Mitotic chromosome staining is an expected exception to an exclusively nucleolar pattern (UniProt Q9BYG3 subcellular location). |
| Strong staining in adipocytes or smooth muscle cells. | These cell types were scored Not detected, so strong signal merits a specificity and detection-background check (HPA: adipocytes and smooth muscle cells). A single unexpected cell does not prove cross-reactivity; compare its compartment and the same-run controls (standard IHC practice). |
| Uniform haze across nuclei, cytoplasm, and extracellular areas. | This obscures the discrete nucleolar pattern and suggests nonspecific staining or detection background (HPA: ubiquitous nucleolar profile; standard IHC practice). Read cell-type intensity only after the background is controlled (standard IHC practice). |
| No detectable signal in colon glandular cells on the test slide. | Colon glandular cells are scored High, making this a useful positive-reference failure (HPA: colon glandular cells). First check the same-run positive section and staining workflow; absence alone cannot establish that NIFK is absent from this specimen (standard IHC practice). |
💡Expected NIFK appearanceCall a result positive when identifiable nuclei show discrete nucleolar chromogenic staining in expected cells, such as High-scored colon glandular cells; strong membrane staining or uniform haze is suspect, while mitotic chromosome staining can be genuine (HPA: colon and nucleolar profile; UniProt Q9BYG3 subcellular location; standard IHC practice).
| Tissue and cell selection | Use a High-scored cell population to judge assay performance, such as colon glandular cells or cerebral cortex neurons (HPA: tissue IHC). Adipocytes and smooth muscle cells provide reported Not detected comparisons, but neither is a universal negative control (HPA: tissue IHC). |
| Interpreting a negative cell | HPA describes ubiquitous nucleolar expression but scores some specified cells Not detected and others Low, including lung macrophages (HPA: tissue IHC). Record the cell type and compartment before treating a faint or absent cell as an assay failure (standard IHC practice). |
| Mitotic cells | Chromosome-associated staining may be appropriate in mitosis because NIFK localizes to mitotic chromosomes with MKI67 (UniProt Q9BYG3 subcellular location). HPA ICC-IF also reports mitotic chromosome localization as supported (HPA: subcellular). |
| IHC evidence strength | HPA rates the overall tissue profile Supported with medium staining–RNA consistency; its listed antibodies have Supported IHC status (HPA: reliability and antibodies). Use the compartment and cell-type pattern together rather than treating a strong brown deposit alone as confirmation (standard IHC practice). |
| IF/ICC Q&A: what should fluorescence show? | Mainly nucleoli and nucleolar rims, with additional mitotic chromosome signal (HPA: subcellular ICC-IF). This is an interpretation reference for IF/ICC; evaluate the paraffin IHC result against the tissue IHC profile (HPA: tissue IHC and subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| High-scored positive cells show no nucleolar signal. | The staining run may have failed, or the chosen cells may be difficult to identify (standard IHC practice). | Check a same-run section containing colon glandular cells, scored High, then review antigen retrieval, primary-antibody application, and detection steps using the validated IHC workflow (HPA: colon; standard IHC practice). |
| Membrane or diffuse cytoplasmic signal dominates. | The compartment conflicts with NIFK's nuclear and nucleolar localization (UniProt Q9BYG3 subcellular location). | Recheck focus and the nuclear counterstain; compare with a High-scored tissue and a primary-omission detection control before scoring the signal as NIFK (HPA: tissue IHC; standard IHC practice). |
| Strong deposits appear in adipocytes or smooth muscle cells. | Those populations are scored Not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; standard IHC practice). | Inspect staining without primary antibody, confirm cell identity, and compare nucleolar localization with a High-scored population on the same run (standard IHC practice; HPA: tissue IHC). |
| The whole section has brown haze. | Excess background can conceal nucleolar deposits (standard IHC practice). | Check reagent-only controls and the validated blocking, washing, primary-antibody dilution, and chromogen timing steps; reassess only after distinct nuclei can be read (standard IHC practice). |
| Only scattered dividing cells show chromosome-associated signal. | Mitotic chromosome localization is documented for NIFK (UniProt Q9BYG3 subcellular location; HPA: subcellular ICC-IF). | Confirm that the stained structures are in mitotic cells, then assess nucleolar staining separately in interphase cells from a High-scored population (UniProt Q9BYG3 subcellular location; HPA: tissue IHC). |
| A negative result is attributed to fixation sensitivity. | The supplied UniProt and HPA records do not report a NIFK-specific fixation effect (UniProt Q9BYG3 record; HPA: tissue IHC). | Document the processing conditions and compare same-run controls before assigning a cause; use antigen retrieval according to the validated IHC workflow (standard IHC practice). |
Experiment evidence
IHC & IF images showing NIFK staining
A04797 · IHCImmunohistochemistry (IHC) analyzes of NIFK (D229) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
P30452 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
P30452 · IHCImmunohistochemistry analysis of paraffin-embedded human brain, using NIFK (Phospho-Thr234) Antibody. The picture on the right is blocked wi
P30452 · IFImmunofluorescence analysis of HUVEC cells, using NIFK (Phospho-Thr234) Antibody. The picture on the right is blocked with the phospho pepti
A30452 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using NIFK Antibody. The picture on the right is blocked with the syn
A30452 · IFImmunofluorescence analysis of HUVEC cells, using NIFK Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000155438-NIFK/tissue/cerebral+cortex#imid_3871344
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000155438-NIFK/tissue/colon#imid_3871278
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000155438-NIFK/tissue/liver#imid_3871287
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000155438-NIFK/tissue/pancreas#imid_3871230
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB015382; green = NIFK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB015382; green = NIFK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NIFK IHC & IF
🧪Run colon first and look for nucleolar staining in glandular cells (HPA: High in colon glandular cells); use smooth muscle as the negative tissue (HPA: Not detected in smooth muscle cells). On the colon slide, adjacent stromal cells with little or no nucleolar staining can serve as provisional internal negative comparators, but their NIFK-negative status is unverified.
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NIFK in A-431, U-251MG, U2OS, PC-3, with annotated localisation: Nucleoli (enhanced), Nucleoli rim (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-matched nonimmune IgG controls for the catalog polyclonal antibody (caption: pAb), plus NIFK-knockout material or immunizing-peptide competition if available. For colon chromogenic IHC, quench endogenous peroxidase and check endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: No NIFK-specific fixation window or fixation effect is reported in the supplied evidence; the A04797 paraffin-section caption does not report a fixative (caption: paraffin-embedded breast carcinoma, fixative unreported). Optimize antigen retrieval empirically: the evidence establishes neither a target-specific retrieval requirement nor that frozen sections or IF are easier. Endogenous peroxidase in colon inflammatory cells may mimic chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for NIFK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
| Nasopharynx | Basal cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NIFK IHC Tips
Troubleshoot NIFK staining in paraffin sections by checking nucleolar localisation, tissue controls and the steps that can obscure or mimic chromogenic signal.
How should I retrieve NIFK when nucleolar staining is weak in paraffin sections?
Use Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Keep section thickness, heating and cooling consistent across the test and control slides so a retrieval difference does not masquerade as a tissue difference (standard IHC practice). Evaluate staining in nucleoli and at nucleolar rims, where NIFK is expected, rather than judging total nuclear colour alone (HPA: subcellular localisation). If signal remains weak, compare a shorter heating time with the page condition and check tissue retention and primary-antibody concentration (standard IHC practice). Record both nucleolar signal and tissue damage before adopting a revised condition (standard IHC practice).
Could fixation explain weak or diffuse NIFK staining?
NIFK-specific fixation sensitivity is unknown because no target-specific fixation comparison is supplied (evidence payload: no NIFK fixation study). The selected antibody image shows staining in paraffin-embedded human breast carcinoma at 1:100, but its caption does not state the fixative (caption: A04797). For chromogenic IHC, compare sections with documented processing histories while holding retrieval, antibody dilution and detection conditions constant (standard IHC practice). Examine morphology and section adhesion alongside nucleolar staining, since damaged sections make a weak signal difficult to interpret (standard IHC practice). Do not assign a fixation cause solely from a change in staining intensity; confirm it with a controlled processing comparison (standard IHC practice).
What staining pattern should count as NIFK in tissue IHC?
Look for discrete nucleolar or nucleolar-rim staining within intact nuclei, the principal NIFK pattern reported by HPA (HPA: subcellular localisation). Chromosome-associated signal can occur in mitotic cells, consistent with NIFK localisation alongside MKI67 during mitosis (UniProt Q9BYG3: subcellular localisation). Assess these compartments at high magnification against the haematoxylin counterstain before counting a cell as positive (standard IHC practice). Diffuse cytoplasmic colour alone does not match the supplied localisation evidence and warrants review of background and detection controls (HPA: subcellular localisation; standard IHC practice). Compare representative fields away from folds and section edges, where chromogen deposits can complicate compartment assignment (standard IHC practice).
How do I troubleshoot possible epitope masking without an antibody epitope map?
No NIFK isoforms are listed, and the supplied record does not identify this antibody’s epitope (UniProt Q9BYG3: isoforms; evidence payload: no epitope map). NIFK contains an RNA recognition motif at residues 45–123 and several annotated modified residues, including phosphoserine 145 and 218 (UniProt Q9BYG3: domains and modified residues). Those annotations do not establish whether any modification affects this antibody’s tissue staining (evidence payload: no epitope or modification-dependence study). If nucleolar signal is lost, vary retrieval exposure in matched sections while keeping antibody and detection settings fixed (standard IHC practice). Treat any apparent modification-dependent pattern as a hypothesis requiring epitope mapping or an appropriate independent control (standard IHC practice).
How can IF help assess a puzzling chromogenic NIFK pattern?
Use IF as a separate localisation check, then judge whether its nucleolar pattern agrees with the paraffin-section IHC result (HPA: subcellular localisation; standard IF/IHC practice). For multiplex IF in colon, pair NIFK with a validated glandular-cell marker and verify each channel independently, since HPA reports high NIFK staining in colonic glandular cells (HPA: colon glandular cells, High; standard IF practice). Choose a far-red NIFK fluorophore when shorter-wavelength tissue autofluorescence obscures signal, and inspect an unstained section (standard IF practice). Because NIFK is nuclear and has no transmembrane segment, permeabilisation must allow antibody access to the nucleus (UniProt Q9BYG3: localisation and topology; standard IF practice). Optimise permeabilisation on IF controls without treating the paraffin IHC caption as IF fixation evidence (caption: A04797; standard IF practice).
What should I check when brown staining obscures NIFK-positive nucleoli?
First check whether the brown product resolves to nucleoli or nucleolar rims in intact nuclei (HPA: subcellular localisation). Include a no-primary control to assess detection-system deposits, and use a peroxidase block before an enzyme-based chromogenic reaction (standard IHC practice). Compare the control with a section receiving the primary antibody under the same DAB development time, counterstain and imaging settings (standard IHC practice). If widespread colour persists, review blocking, antibody concentration and wash stringency one variable at a time (standard IHC practice). Exclude folds, damaged edges and necrotic areas when judging whether background has improved, because these regions can retain misleading chromogen (standard IHC practice).
How should I score NIFK staining across heterogeneous tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define positivity by nucleolar or nucleolar-rim staining in intact, identifiable cells before examining group differences (HPA: subcellular localisation; standard IHC practice). Report the percentage of positive cells or an H-score using fixed intensity categories, with the same exposure and scoring rules for every section (standard IHC practice). For spatial comparisons, count positive cells per mm² and normalise to the number of evaluable cells or tissue area in the same compartment (standard IHC practice). Score glandular cells separately from other populations in colon, where HPA reports high glandular-cell staining (HPA: colon glandular cells, High). Exclude necrosis, folds and section edges using prespecified criteria, and report how many cells or fields contributed to each score (standard IHC practice).
When should an apparent NIFK-positive cell be considered an artefact?
A convincing positive shows nucleolar or nucleolar-rim signal in an intact nucleus; mitotic chromosome staining is also compatible with the reported localisation (HPA: subcellular localisation; UniProt Q9BYG3: mitotic localisation). Check cell identity against the tissue compartment: HPA reports high staining in colonic glandular cells but no detection in adipocytes (HPA: colon glandular cells, High; adipose adipocytes, Not detected). Isolated cytoplasmic colour, staining confined to a section edge or signal over necrosis needs closer scrutiny (HPA: subcellular localisation; standard IHC practice). Compare suspect areas with a no-primary control to identify deposits or endogenous enzyme activity (standard IHC practice). Interpret modest tissue differences cautiously because HPA rates its tissue IHC evidence Supported with medium staining-to-RNA consistency (HPA: reliability).
Boster reagents
Best NIFK / MKI67 FHA domain-interacting nucleolar phosphoprotein IHC Antibodies
Anti-NIFK antibodies have IHC images from paraffin-embedded human breast carcinoma and brain, plus IF images from HUVEC cells; catalog reactivity includes human, mouse, and, for A04797, rat (catalog images and reactivity).
Real IHC data
Anti-NIFK (D229) Antibody
Cat # A04797
Real IHC data
Anti-Phospho NIFK (Thr234) Antibody
Cat # P30452
Real IHC data
Anti-NIFK Antibody
Cat # A30452
A04797 shows IHC in paraffin-embedded human breast carcinoma and lists IF, with human, mouse, and rat reactivity (A04797 IHC caption and catalog). P30452 shows phospho-Thr234 IHC in paraffin-embedded human brain and IF in HUVEC cells (P30452 image captions); A30452 shows IHC in paraffin-embedded human brain and IF in HUVEC cells (A30452 image captions).
Which to pick: For tissue IHC, choose A04797 for the demonstrated breast carcinoma sample, A30452 for NIFK in the demonstrated brain sample, or P30452 when the phospho-Thr234 target is required (each SKU’s IHC caption). For IF/ICC, A30452 and P30452 are listed for both applications, are polyclonal, and have their own HUVEC IF images (catalog applications, dilution data, and IF captions). A04797 is the cross-species choice when rat reactivity matters (catalog reactivity); the fixative is unreported in all three paraffin-section IHC captions.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BYG3 (MK67I_HUMAN, MKI67 FHA domain-interacting nucleolar phosphoprotein).
- Human Protein Atlas. NIFK tissue IHC expression (reliability: Supported).
- Human Protein Atlas. NIFK subcellular location (ICC-IF): Mainly localized to the nucleoli and nucleoli rim. In addition localized to the mitotic chromosome..
- Human Protein Atlas. NIFK antibody validation summary (5 antibodies).
- NIFK as a potential prognostic biomarker in colorectal cancer correlating with immune infiltrates. Medicine 2023 — PMC10553176.
- PubMed PMID:11342549 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NIFK (MKI67 FHA domain-interacting nucleolar phosphoprotein). Expected localisation Nucleolar staining in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A04797 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A04797); 2. Fixation: Image fixative and duration unreported (datasheet A04797); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-NIFK, 1:50-1:200 (datasheet A04797); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across tissue samples (standard IHC practice; not target-specific); Caveat: Antibody staining has medium RNA concordance (HPA tissue IHC); Isoform / epitope: No isoforms reported; no membrane topology (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt Q9BYG3, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoli and nucleoli rim. In addition…, Datasheet A04797, PMC10553176. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NIFK/immunohistochemistry-ihc-guide.